失去phytochromes的拟南芥生活周期

Arabidopsis thaliana life without phytochromes Bárbara Strassera,1, Maximiliano Sánchez-Lamasa,1, Marcelo J. Yanovskyb, Jorge J. Casalb, and Pablo D. Cerdána,2 aFundación Instituto Leloir, Instituto de Investigaciones Bioquímicas de Buenos Aires–Consejo Nacional de Investigaciones Científicas y Técnicas and Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, C1405BWE Buenos Aires, Argentina; and bInstituto de Investigaciones Fisiológicas y Ecológicas Vinculadas a la Agricultura, Facultad de Agronomía, Universidad de Buenos Aires and Consejo Nacional de Investigaciones Científicas y Técnicas, 1417 Buenos Aires, Argentina Edited* by Joanne Chory, The Salk Institute for Biological Studies, La Jolla, CA, and approved January 22, 2010 (received for review September 11, 2009) Plants use light as a source of energy for photosynthesis and as a source of environmental information perceived by photorecep- tors. Testing whether plants can complete their cycle if light provides energy but no information about the environment requires a plant devoid of phytochromes because all photo- synthetically active wavelengths activate phytochromes. Produc- ing such a quintuple mutant of Arabidopsis thaliana has been challenging, but we were able to obtain it in the flowering locus T (ft) mutant background. The quintuple phytochrome mutant does not germinate in the FT background, but it germinates to some extent in the ft background. If germination problems are bypassed by the addition of gibberellins, the seedlings of the quintuple phytochrome mutant exposed to red light produce chlorophyll, indicating that phytochromes are not the sole red- light photoreceptors, but they become developmentally arrested shortly after the cotyledon stage. Blue light bypasses this block- age, rejecting the long-standing idea that the blue-light receptors cryptochromes cannot operate without phytochromes. After growth under white light, returning the quintuple phytochrome mutant to red light resulted in rapid senescence of already expanded leaves and severely impaired expansion of new leaves. We conclude that Arabidopsis development is stalled at several points in the presence of light suitable for photosynthesis but providing no photomorphogenic signal. Clock | cryptochrome | germination | photosynthesis Plants use light as a source of energy for photosynthesis and asa source of information about their surrounding environment. Phytochromes, cryptochromes, phototropins, and the zeitlupe family of photoreceptors capture the signals of the environment that provide spatial and temporal information and control growth and development (1). Phytochromes have absorbance maxima in the red (660 nm) and far-red light (730 nm). They are synthesized as Pr (the inactive form) that is converted by red light to the active form, Pfr. This reaction is reversible by far-red light, which converts Pfr back to Pr. Five phytochrome apoprotein genes are present in the reference plant Arabidopsis thaliana (PHYA–PHYE) (1), each with partially overlapping functions (2). Conversely, only three phytochrome genes are present in rice (3). Phytochromes bear a covalently attached linear tetrapyrrol chromophore, the phytochromobilin, which undergoes a cis-trans isomerization when photoconverted (4). Phytochromes promote germination of sensitized Arabidopsis seeds even after a brief exposure to very low fluence of light, which may occur during disturbance of the soil surface (5). After this transient exposure to light, the seed germinates in darkness, under the soil surface, and uses seed reserves to grow against the gravitropic vector; the cotyledons remain closed and folded down to prevent damage to the apical meristem. Once the seedling reaches the soil surface it undergoes a light-triggered developmental transition, termed de-etiolation; where hypocotyl growth is arrested, the cotyledons unfold, open and turn green, establishing the photomorphogenic pattern of development (6). The triple phyA phyB phyC mutant of rice lacks inhibition of coleoptile growth, detectable synthesis of chlorophyll, and changes in gene expression under continuous red light, indicating that in grasses, phytochromes are the sole photoreceptors for red and far-red light during de-etiolation (3). De-etiolation is also promoted by the UV-A and blue-light photoreceptors cryptochromes (7), which interact with phyto- chromes in the control of this transition (8). Based on classic photobiological experiments, Hans Mohr (9) had proposed that the sole action of blue light perceived by specific photoreceptors (now identified as cryptochromes) was to amplify the response to Pfr (i.e., cryptochrome action requires Pfr). Under suboptimal light input conditions, cryptochrome action requires phyB activity (10), and under those conditions, phyB Pfr has recently been shown to act downstream of cryptochrome as predicted by Mohr’s model (11). Under prolonged exposures to blue light, cryptochromes operate independently of phyA and phyB (12), but they could depend on other members of the phytochrome family (8, 12). Because phytochromes also absorb blue light, a definitive test for this classic proposition is impossible without the quintuple phytochrome mutant (8, 12). Green vegetation canopies lower the red to far-red ratio of the light. This reduces the activity of light-stable phytochromes such as phyB but enhances the activity of phyA (13). The hypocotyl- growth response to red/far-red ratio represents a balance between these actions. Photosystem I and even photosystem II extend their activity to the far-red region of the spectrum (14). The latter could be negligible under a strong background of light between 400 and 700 nm, but it could be important for the responses to shade when the proportion of far-red light is high (14). Because different phytochromes have opposing effects, the onlyway to test the actual contribution of photosynthetic reactions to the growth response to far-red light is to use plants without phytochrome. Most organisms, from bacteria to humans, have an internal clock that allows them to synchronize daily and seasonal rhythms in physiological processes with periodic environmental changes. To maintain an anticipatory function throughout the year, cir- cadian clocks must be adjusted daily. Such entrainment is effected, in part, through pathways that signal information from light–dark transitions to the clock. Genetic evidence indicates that phytochromes, cryptochromes, and members of the zeitlupe family of photoreceptors control the circadian oscillator in Ara- bidopsis plants (15). In mammals, cryptochromes are required to sustain circadian rhythms even in complete darkness (16). In contrast to what is observed in mammals, cryptochromes are not essential for clock function in plants (17, 18). Interestingly, most circadian clock mutants show defective developmental responses to red light (15). It is unknown whether the latter is just a consequence of circadian modulation of phytochrome Author contributions: M.J.Y., J.J.C., and P.D.C. designed research; B.S., M.S.-L., M.J.Y., and P.D.C. performed research; M.J.Y., J.J.C., and P.D.C. analyzed data; and M.J.Y., J.J.C., and P.D.C. wrote the paper. The authors declare no conflict of interest. *This Direct Submission article had a prearranged editor. 1B.S. and M.S.-L. contributed equally to this work. 2To whom correspondence should be addressed. E-mail: pcerdan@leloir.org.ar. This article contains supporting information online at www.pnas.org/cgi/content/full/ 0910446107/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.0910446107 PNAS Early Edition | 1 of 6 PL A N T BI O LO G Y signaling or reflects an involvement of phytochromes in the core mechanism underlying circadian rhythms in plants. Here we report the isolation of the Arabidopsis quintuple phytochrome mutant. We show that in contrast to the rice triple mutant devoid of phytochrome, the quintuple phytochrome mutant of Arabidopsis partially greens under red light. We also demonstrate that both blue light-induced photomorphogenesis and phototropism and the circadian clock operate in the absence of phytochromes. However, the photomorphogenic signal is crucial, and in its absence photosynthetically active radiation is not sufficient to sustain development. Results Isolation of the Quintuple Phytochrome Mutant in the ft Background. Two alternative strategies could be used to obtain plants without active phytochromes. One is to combine mutations to block phytochrome chromophore biosynthesis. This approach has not been successful because of the close linkage of some gene family members (19) and the leakiness of mutants at some loci (20). The second strategy is to combine alleles of the five PHY apo- protein genes. Our initial attempts to isolate the quintuple phytochrome apoprotein mutant from segregating populations were unsuccessful. However, in the progeny of a phyA phyC phyD phyE ft mutant that was heterozygous for phyB, we noted that a few plants (<0.5%) were unable to de-etiolate under red light, which is perceived mainly by phyB (2). We transferred these seedlings to white light and confirmed that they were indeed the quintuple phytochrome mutants in the ft background. The Quintuple Phytochrome Mutant Depends on Exogenous GAs for Seed Germination.The involvement of individual phytochromes in seed germination is well established (21), so we tested the ability of the quintuple phytochrome mutant to germinate under dif- ferent light conditions. Seeds were stratified for 3 days and then incubated at 23 °C under white, red, far-red, or blue light, or kept in darkness after sowing. The germination rate of the quintuple phytochrome mutant was very low compared to the other control genotypes because none of the light treatments increased its germination rates above dark controls (Fig. 1A), even at high fluencies of red or blue light (Fig. S1). All of the other genotypes used here showed high percentages of germination at least under some light conditions (Fig. 1A and Fig. S1). The low germination frequency of the quintuple phytochrome mutant could be the consequence of irreversible defects on seed or embryo development or the induction of a dormant state that could not be reversed because of the absence of phytochrome. Because phytochromes induce germination by increasing GA biosynthesis (22, 23), we tested the ability of GA to restore germination. Despite the fact that the germination rate of the quintuple phytochrome mutant was very low, we could induce normal germination levels by adding GA4, but not GA3 (Fig. 1B and Fig. S2), showing that seeds are viable but remain dormant in the absence of the light signal provided by phytochromes. The promotion of seed germination by low temperatures occurs at least partially by inducing GA biosynthesis during seed stratification (imbibition at low temperatures) (24). Low tem- peratures can substitute for light exposure in germination assays (24), but even after a far-red light treatment it is technically phyA phyB phyC phyD phyE ft ft WT 0 2010 30 Stratification time (days) C -GA GA3 GA4 0 20 40 60 80 100 % G er m in a tio n B phyA phyC phyD phyE ft phyB phyD phyE ft 0 20 40 60 80 white blue far-red red dark ftWT phyA phyB phyA phyB phyA phyB phyC phyD phyE ft 100 % G er m in a tio n A Fig. 1. Germination of the quintuple phytochromemutant is not light responsive and requires exogenous GA. (A) Seeds were plated onMurashige and Skoog (MS) salts agar, stratified for 3 days at 4 °C, and incubated for 5days under different light regimes at 23 °C before counting germinated seeds (radicle emergence). Light conditions:white light, 80μmolm−2 s−1; red light, 10 μmolm−2 s−1; far-red light, 60 μmolm−2 s−1; blue light, 10 μmolm−2 s−1; anddarkness. Data are averages ± SE of six or three (far-red) independent experiments with 50 seeds each. None of the light treatments promoted germination of the quintuple phytochrome mutant (one-wayANOVA,P=0.43). (B) Seeds of thequintuplephytochromemutantwere sownonmoistenedfilter paper containing 100μMGA3,GA4 (Sigma), or no hormone and stratified for 3 days at 4 °C before the induction of germination at 23 °C under red light. Data scored 5 days later are averages ± SE of five independently collected seed pools (50 seeds each). One-way ANOVA followed by Bonferroni tests indicated significant differences between the control and +GA4 (P < 0.01). (C) Seeds of the WT, ft, and phyA phyB phyC phyD phyE ftmutants were sown on agar containing MS salts and stratified at 4 °C for the times indicated on the abscissa. Germination was induced and scored as in (B). Data are averages ± SE of five independently collected seed pools for the quintuple phytochrome mutant and two for the WT and ft controls. A t test indicates that the quintuple phytochrome mutant responded to stratification (P < 0.001). 2 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.0910446107 Strasser et al. impossible to remove all active phytochrome (i.e., to convert all Pfr back to Pr), because far-red light can produce low levels of Pfr due to the overlapping Pr and Pfr spectra (1). Therefore, we tested whether low temperatures could induce germination in the quintuple phytochrome mutant. Seeds were incubated in the dark at 4 °C for periods of variable duration and then moved to 23 °C to score germination percentages 5 days later (Fig. 1C). Stratification improved germination but did not restore the germination potential of the quintuple phytochrome mutants to those observed with added GA (Fig. 1 B and C, and Fig. S2). GA addition to the nongerminating seeds after ending the experi- ment was still effective in inducing germination. Cryptochromes Promote De-Etiolation in the Absence of Phytochromes. The model proposed by Hans Mohr (9) states that cryptochromes require at least some level of Pfr to effectively induce a response. Therefore, we decided to investigate the hypocotyl elongation response to blue light—a cryptochrome mediated response (1)—in the phytochrome-less mutant. The phyA phyB phyC phyD phyE ftmutant responded well to blue light, showing that cryptochromes do not require phytochromes to trigger the inhibition of hypocotyl elongation under blue light (Fig. 2A). Conversely, the quintuple phytochrome mutant was not responsive to red light at this stage as expected based on previous observations showing that inhibition of hypocotyl growth by red light is already absent in the phyA phyB double mutant (25). The latter is also consistent with a recent report showing that the triple phyA phyB phyCmutant of rice, a species that only has these three phytochromes, fails to respond to red light (3). Cotyledon opening and greening were also triggered by blue light (Fig. 2B). The hypocotyl phototropic response was present in the quintuple phytochrome mutant, indicating that phototropins can still func- tion in the absence of phytochrome (Fig. S3). Chlorophyll Synthesis in the Absence of Phytochrome.One of the last steps in chlorophyll synthesis, the conversion of proto- chlorophyllide into chlorophyllide, is catalyzed by the light-driven enzyme protochlorophyllide oxidoreductase. Although this enzyme is directly activated by light (26), the phyA phyB phyC rice mutant lacks detectable chlorophyll levels. Conversely, we were able to measure the conversion of protochlorophyllide into chlorophyllide after a red light pulse, detected as a decrease of emission at 635 nm (protochlorophyllide) and increase of emission at 670 nm (chlorophyllide), in the quintuple phytochrome mutant of Arabidopsis (Fig. 2H). These results show that Arabidopsis plants devoid of phytochromes are not totally blind to red light. Isolation of the Quintuple phyA phyB phyC phyD phyE Mutant in the FT Background. With the knowledge acquired about the quintuple phytochromemutant, we tried to isolate the correspondingmutant in the FT (WT) background. We used a phyA phyD phyE mutant population segregating for phyB and phyC, induced to germinate with GA. Etiolated seedlings were selected under red light, allowed to de-etiolate in white light, transplanted to soil, and genotyped for phyB and phyC. Under white light, the phyA phyB phyC phyD phyE mutants were tiny plants as compared with the isogenic line in the ftbackground andonly formed a couple of small siliques (Fig. 2F). None of the seeds obtained germinated in the absence of GA (at least 200 seeds were tested). These results underscore the advantages of having used the ft background, which was recently shown to improve germination (28). The ft mutation caused a delay in flowering time in the quintuple phytochrome mutant background (Fig. 2F) allowing sufficient seed production. Therefore, we decided to continue our work with the genotypes in the ftmutant background that also allow a longer vegetative phase, useful for the subsequent experiments. The Quintuple Phytochrome Mutant Lacks Growth Responses to Red/ Far-Red Ratio.Green leaves reflect and transmit far-red light more efficiently than red light and, therefore, neighbor plants lower the level of active phytochrome and induce shade-avoidance reac- tions, which typically include accelerated stem growth (29). In our conditions, the addition of far-red light lowered the red/far-red ratio and promoted hypocotyl growth in the phyA phyC phyD phyE ftmutant, where only phyB is present (Fig. 2C). Conversely, in the phyB phyC phyD phyE ftmutant, where phyA is the only remaining phytochrome, supplementary far-red light reduced hypocotyl growth because of the high-irradiance response mediated by phyA (Fig. 2C). In the WT and ft mutant, supplementary far-red light caused some reduction of hypocotyl growth indicating that the high-irradiance response (30) dominates over the shade- avoidance response at this early stage of the life of the seedling (Fig. 2C; see also ref. 31). Red/far-red reversibility is the classic signature of phytochrome activity, and no response to the red/far- red ratio was observed in the quintuple phytochrome mutant (Fig. 2C). Developmental Arrest of the Quintuple Phytochrome Mutant Under Red Light. To test whether photosynthetic light unable to provide photomorphogenic signals is enough to sustain plant develop- ment, we cultivated the phyA phyB phyC phyD phyE mutant in the ft background under continuous red light. All of the quin- tuple phytochrome mutants that germinated under red light (at least 10 plants in two independent experiments) did not develop beyond the cotyledon stage. In subsequent experiments we decided to include sucrose in the media, to increase the lifetime of the plant and the possibility of a light-triggered development. We avoided the contact of sucrose with the aerial tissues (Fig. S4) because the latter provides a morphogenic signal sufficient to complete the life cycle in the dark (32). In some seedlings, sucrose promoted root growth and some additional development of aerial tissues. However, most seedlings (at least 20 plants in two independent experiments) did not develop more than a long hypocotyl and a barely expanded pair of cotyledons; some seedlings showed stem extension above the cotyledonary node and some rudimentary unexpanded leaves (Fig. 2D). We were able to detect chlorophyll in the quintuple mutant even after 8 days under red light (Fig. 2G and Fig. S5). However, seedlings ended up turning brownish, and development became arrested after 6 to 8 weeks (Fig. 2 B and D). Blue light bypassed this block, because the quintuple phytochrome mutant grown under blue light was capable of vegetative development and flowering (Fig. 2E). As controls, the phyA phyB double mutant or the phyB phyC phyD phyE ft quadruple mutant bearing only active phyA were able to develop and flower under red light alone (Fig. 2E). Because under red light Arabidopsis phyA has a half-life of only 30 min (33), the latter demonstrates how little phytochrome is e